Hepatocellular carcinoma (HCC) is a significant and deadly cancer in the world. Chronic hepatitis virus infection is the most significant risk factor for HCC development, especially among populations in Asia, Africa and the Amazon Basin. Importantly, there is a significant sex disparity among the HCC patients infected with hepatitis B virus (HBV) with male to female ratio as high as 6:1, depending on geographical locations. Currently, the exact etiology of such male dominance in the disease is uncertain. Recent studies suggest that the expression of the viral oncogene, HBx, on HBV genome is regulated by androgen, the male hormone, and androgen receptor (AR). HBx, in term, can activate AR, thereby generating a positive feedback loop for its own expression and oncogenic functions. Studies of another oncogene on the Y chromosome, testis-specific protein Y encoded (TSPY), suggest that TSPY serves as a co-activator for AR transactivation and stabilizer for HBx protein and could exert significant male dominant effects on HBx expression and oncogenesis. TSPY has specialized as a male germ cell factor involved in stem cell proliferation and meiotic division, but functions as an oncogene when ectopically expressed in incompatible somatic cells. Interesting, there is a X-homologue of TSPY, TSPX, which possesses contrasting properties and acts as a tumor suppressor. TSPX co-represses AR transactivation and binds but promotes HBx proteasomal degradation. After it diverged with TSPY, TSPX maintains the housekeeping functions in modulating proper cell cycle progression, promoting viral protein degradation and repressing viral gene expression. TSPY disrupts TSPX functions, and promotes viral oncogenesis in populations chronically infected with HBV. TSPY could exacerbate HBx oncogenic functions by exaggerating the AR transactivation of HBx gene and maintaining HBx protein stability. In this exploratory project, we plan to address the roles of TSPY and TSPX in HBx protein stability and modulation AR transactivation. First, we will elucidate the mechanisms involved in TSPX promotion of HBx degradation in the ubiquitin dependent and independent pathways. We will define the domains responsible for TSPY and TSPX interactions with and modulation of HBx proteasomal degradation. We will examine the postulation that the domains in TSPX, which are absent in TSPY, could serve tethering and enhancer functions between HBx and the proteasomal machinery. Second, we plan to evaluate three putative androgen response elements (AREs) at the distal Enhancer I position upstream of the HBx gene promoter in the AR transactivation by confirming TSPX and TSPY interactions with AR and delineating the critical domains important for TSPY and TSPX co- activation and co-repression respectively with AR on HBx transactivation. We will examine TSPY functions on HBx promoter activities. Understanding how TSPY disrupts TSPX functions and exacerbates HBx expression and protein stability will shed critical insights on the potential contributions of this pair of homologous oncogene and tumor suppressor gene in the pathogenesis of male dominance in HBV-related hepatocarcinogenesis. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinoma is a prevalent and deadly liver cancer in the world, particularly among populations chronically infected with hepatitis viruses. Significantly, there is a critical gender disparity favoring men among hepatocellular carcinoma patients infected with hepatitis B virus. The reason for such male preference in the disease process is unknown. Recent studies have identified an oncogene (cancer gene), designated as TSPY on the human Y (male-only) chromosome that affects the expression and protein stability of the hepatitis B viral cancer gene, called HBx. Importantly, there is a homologue of TSPY, designated as TSPX, on the X chromosome, which possesses contrasting properties on HBx, and functions as a tumor suppressor. The proposed research is designed to elucidate the functions of this pair of homologous oncogene and tumor suppressor gene on HBx and male dominance in hepatocellular carcinoma development in hepatitis B virus infected populations.